Certain T-even and T-odd bacterial viruses contain genetic information for the synthesis of tRNAs: this information is expressed following infection of Escherichia coli. For T5 bacteriophage, a full complement of 20 tRNA species, including isoacceptor tRNAs, have been identified and their physical map position has been located in T5 DNA. The tRNA gene map was constructed by tRNA DNA hybridization techniques using a number of T5 DNA deletion mutants in which the size and location of the deleted DNA segments were accurately determined by heteroduplex-electron microscopy mapping. The current map shows the presence of four separate tRNA regions clustered in the C segment of T5 DNA, except for the tRNAArg gene which is located at the left end of the D segment. Our immediate objective is to refine this map so that the individual tRNA genes in each cluster can be precisely positioned along the heavy T5 DNA strand. After positioning each tRNA gene, our long term objective is to study the regulatory mechanism for phage tRNA transcription, in vitro, using appropriate T5 DNA fragments containing all or a portion of the mapped tRNA genes. The refinement of the tRNA gene map is currently underway using restriction endonucleases; further refinement will be carried out by DNA sequence analysis in the tRNA gene region of T5 DNA.